The major hypothesis examined by the University of Virginia Udall Parkinson's Center is that mitochondrial genome mutations contribute substantially to the pathogenesis of sporadically occurring Parkinson's disease (PD), which is the form afflicting >95% of PD patients. During the first funding period, substantial progress was made in demonstrating with platelet mitochondrial DNA (mtDNA)-derived cybrids that expression of PD mitochondrial genomes reproduces multiple essential pathobiological features of PD. Most dramatically, PD cybrids spontaneously generate Lewy inclusion bodies that are indistinguishable biochemically and morphologically from those present in PD brain. Difficulties in acceptance of the mitochondrial genomic contribution to PD derive in part from the lack of specific mtDNA mutations yet defined in PD brain, concerns about the relevance of PD platelet mtDNA to events in brain neurons, inability to provide direct proof of causality of mtDNA mutations and lack of understanding of how bioenergetic deficits and proteasomal dysfunction can arise in PD from mtDNA mutations. All of these concerns are systematically and comprehensively addressed in this renewal application. Dr. Bennett's project utilizes novel technologies created at UVA that allow the insertion into mitochondria and expression in cells of human mitochondrial genomes from any source including postmortem brain. Cell lines expressing mtDNA from well characterized PD brains will be created and analyzed phenotypically, allowing a direct test of causality. Mutations can also be created by mutagenesis and expressed in cells, directly demonstrating pathogenecity of mtDNA. Dr. Capaldi's project involves a collaboration with the University of Oregon to provide a detailed proteomic analysis of complex I macromolecular assembly and oxidative damage in PD brain mitochondria compared to mitochondria from cells that express mtDNA from the same PD brains. Project 3 will uncover mechanisms of bioenergetic complex I impairment arising from PD mtDNA mutations. Dr. Trimmer's project examines the appearance of proteasomal impairment in individual cells of PD cybrids and cell lines created from PD brain, and defines how proteasomal impairment relates to development of pathogenic protein aggregates. This project specifically links PD mtDNA expression to neuronal PD pathobiology. All projects are supported by two Cores. The Cell Culture Core provides centralized culturing, characterization and archiving of all cell lines. The Administrative Core provides centralized fiscal oversight and supports regular weekly Center meeting and the annual visits of the External Advisory Committee.